Cellulose films for screening

ABSTRACT

The invention relates to a cellulose film comprising microfibrillated cellulose and to the use of it for screening of a biological compound and nucleic acids encoding a biological compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119 of Danishapplication PA 1999 01414 filed on Oct. 1, 1999 and U.S. provisionalapplication No. 60/157,912 filed on Oct. 6, 1999, the contents of whichare fully incorporated herein by reference.

TECHNICAL FIELD

This invention relates to cellulose films and to methods for their usefor identifying or screening actives such as biological compounds ornucleic acid sequences encoding such. Also the invention relatesbiological compounds found or identified by these methods and to methodsof producing biological compounds identified.

BACKGROUND

The art of identifying useful biological compounds in unknown samples orcompositions, such as enzymes, encompasses disclosures such as WO99/34011 disclosing use of textile test swatches for identifyingenzymes. EP 454 046 B1 discloses a test slide for detecting the presenceof micro-organisms, their enzymes and metabolites. JP 49060289 Adiscloses an enzyme activity test disk for detection of enzymeactivities in the digestive tract.

Bacterial cellulose is described e.g. in disclosures such as U.S. Pat.No. 4,863,565; WO 93/11182 and U.S. Pat. No. 4,861,427.

JP10-95803 discloses bacterial cellulose coatings e.g. for paper.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows degradation of bacterial Cellulose I and Cellulose III_(I)by H. insolens complex.

FIG. 2 shows degradation of bacterial Cellulose I and Cellulose III_(I)by EG V (A) and EG VI (B)

FIG. 3 shows release of DTAF by digestion by H. insolens complex oflabelled bacterial cellulose I as a function of grafting conditions.

FIG. 4 shows release of DTAF by digestion by H. insolens complex oflabelled bacterial cellulose I as a function of grafting steps using 30mg of DTAF in 0.2 N NaOH.

FIG. 5 shows release of DTAF by digestion by EG VI of labelled bacterialcellulose I as a function of grafting steps using 30 mg of DTAF in 0.2 NNaOH.

FIG. 6 shows release of DTAF by digestion by H. insolens complex oflabelled bacterial cellulose I as a function of grafting steps using 60mg of DTAF in 0.2 N NaOH.

FIG. 7 shows release of DTAF by digestion by EG VI of labelled bacterialcellulose I as a function of grafting steps using 60 mg of DTAF in 0.2 NNaOH.

FIG. 8 shows release of DTAF by digestion by H. insolens complex oflabelled bacterial cellulose III_(I) as a function of grafting stepsusing 60 mg of DTAF in 0.2 N NaOH.

FIG. 9 shows release of DTAF by digestion by EG VI of labelled bacterialcellulose III_(I) as a function of grafting steps using 60 mg of DTAF in0.2 N NaOH.

FIG. 10 shows release of DTAF by digestion by active EG VI and inactivemutant EG VI of labelled bacterial cellulose III_(I) as a function ofincubation time.

FIG. 11 shows release of DTAF by digestion by EG V of labelled cottoncellulose I as a function of grafting steps using 60 mg of DTAF in 0.2 NNaOH.

FIG. 12 shows release of DTAF by digestion by EG VI of labelled cottoncellulose I as a function of grafting steps using 60 mg of DTAF in 0.2 NNaOH.

SUMMARY OF THE INVENTION

The present invention relates to a method for screening of an activesuch as a biological compound or a nucleic acid sequence encoding abiological compound using a cellulose film comprising microfibrillatedcellulose. Specifically the invention provides a method for screening oridentifying a an active, preferably a biological compound, comprisingcontacting a sample containing the active with a cellulose filmcomprising microfibrillated cellulose and detecting an interactionbetween the cellulose film and the active.

The invention also relates to cellulose films and processes for theirmanufacture which are suitable for the screening method. Specificallythe invention provides a cellulose film comprising microfibrillatedcellulose, wherein the film further comprises a substance attached tothe microfibrillated cellulose.

Further the invention relates to test containers comprising a cellulosefilm and processes for their manufacture, which are suitable forcarrying out the screening process. Specifically the invention providesa container, preferably having a volume of less than 10 ml, comprisingat least one surface coated with a cellulose film.

Still further, the invention relates to an active, preferably abiological compound identified by the screening method as well asprocesses for their manufacture. Specifically the invention provides anactive, preferably a biological compound and/or a nucleic acid sequenceencoding a biological compound identified by the screening process

DETAILED DESCRIPTION OF THE INVENTION

One object of the present invention is to provide improved methods forfinding new cleaning materials, such as enzymes. In finding newmaterials suitable for cleaning e.g. cellulose containing fabrics onemay chooses to test the new materials on real fabrics to determine ifthey possess any cleaning properties. However, this approach isundesirable because of the slowness and limited capacity of suchmethods. Accordingly another object of the invention is to provideimproved methods capable of testing large numbers of potentialcandidates at a considerable speed. Further objects are to providemethods which can be carried out on small samples and which may beeasily automated.

Definitions

The term “microfibrillated cellulose” as used herein is to be understoodas isolated and purified cellulose fibres recovered from a source in aprocess preserving the original cellulose filamentous structure.Microfibrillated cellulose will hereafter be denoted “MFC”. Alsoencompassed by the this term are cellulose fibres, which after isolationand purification has undergone chemical treatment changing the internalstructure and/or arrangement of the fibres. Consequently the termmicrofibrillated cellulose encompass purified and isolated cellulosefrom microorganisms such as bacterial cellulose (hereinafter denoted“BC”).

In context of the invention, the term “nucleic acid source” is to beunderstood as any DNA, RNA or cDNA material or material comprising DNA,RNA or cDNA.

In the context of the invention, the term expression system is to beunderstood as a system enabling transcription of a nucleic acid sequenceand translation into the synthesis of the corresponding biologicalcompound. The expression system may be a cell or an in vitro system.

In the context of the invention, the term gene library is to beunderstood as fragments of DNA or cDNA derived from a nucleic acidsource.

In the context of the invention, the term “host cell” is to beunderstood as a cell, which may host and may express an inserted DNA orcDNA fragment from a gene library.

In the context of the invention, the term “transformant” or “transformedhost cell” is to be understood as a host cell in which a DNA or a cDNAfragment from a gene library has been inserted.

In the context of the invention, the term “clone” is to be understood asa copy of a cell or a transformed host cell.

The term “active” as used herein is to be understood any compound or amixture of compounds, which perform a measurable interaction with acellulose film and/or any substance incorporated in or associated to acellulose film.

Microfibrillated cellulose

The cellulose film of the invention comprises MFC. We have found thatsuch cellulose films mimics cellulose containing textile surprisinglywell and may advantageously substitute such textile or fabric whenscreening for actives, preferably biological compounds, interacting withcellulose in textile or substances present on a textile surface. This isan important aspect because when searching for new cleaning agents e.g.biological compounds such as enzymes, and testing their effect on acellulose film mimicking a real textile it is more likely that foundcandidates will also work well on real textile. Choosing more artificialtest conditions, however, may generate a large number of falsecandidates in the screening, i.e. enzymes may be found which works wellunder artificial conditions, but will perform poorly on real textile.

MFC also possesses an enhanced accessibility towards e.g. cellulaseenzymes which may reacts more readily with MFC than with cellulose whichhas not been microfibrillated. The enhanced accessibility of MFC alsomeans the MFC is easier penetrated by water. The enhanced accessibilityfurther means that the MFC may be easier modified e.g. by reactingcompounds onto the MFC by e.g. esterification, etherification,sulfonation, phosporylation and/or carboxylation.

Accordingly a cellulose film of the invention may used to identifycleaning agent, such as enzymes, which will also have good cleaningproperties

Moreover an important feature of the cellulose film of the invention isthat it is possible to prepare such cellulose film in very smallcontainers, such as wells in a conventional micro plate. Especially formicro plates containing very small wells such as 96, 384 or 1536 wellplates with corresponding well volumes of 320 μl, 160 μl, and 14 μl,respectively, it is very difficult to use pieces of real textile.

MFC is a form of expanded high volume cellulose, in which cellulosefibres are opened up and unraveled to expose smaller fibrils andmicrofibrils. The fibrils of the MFC in a film of the invention have anaverage length of about at least 10 μm, preferably about at least 50 μm,most preferably about at least 100 μm. However a preferred averagelength of the fibrils is less than about 500 μl, more preferably lessthan about 300 μl, most preferably less than about 200 μl. The averagewidth of the fibrils are between about 50-200 nm, preferably about75-150 nm, most preferably about 80-120 nm. Each fibril consists of abundle of microfibrils. The microfibrils in the fibrils have an averagethickness of about 2-20 nm preferably about 5 nm and each fibrilcontains a bundle of about 50-100 microfibrils. The isolated andpurified fibrils are surprisingly long. In the microfibrils the nativeCellulose I internal structure is preferably retained, so that thepolymeric chains of glucose monomers constituting each cellulose chainare arranged parallel to each other. However, internal structuresobtained by chemical modification of the original structure, by methodsknown to the art, are also preferred such as a Cellulose II structure inwhich the cellulose chains are arranged antiparallel to each other or aCellulose III structure in which the hydrogen bonding of the Cellulose Istructure is altered or a Cellulose IV structure.

Sources of Cellulose

The MFC comprised in the film of the invention may be obtained from anysuitable source. Such as microorganisms producing cellulose or fromplants such as wood (e.g. soft wood or pulped soft wood), cotton, straw,jute, grasses, tunicate or cereals such as bran. However, a source ispreferred in which the cellulose in the source is available in way sothat MFC may be isolated and purified in a way to preserve long fibrilsor microfibrils. Accordingly preferred cellulose sources aremicroorganisms and cotton. Preferred microbial cellulose is bacterialcellulose. Bacterial cellulose contains very long cellulose chainsand/or fibers and has shown very good film forming properties. Suchbacterial cellulose is also commercially available, e.g. from theproduct Nata de Coco, which is a fermented product of coconut milk, fromFujico Company, Kobe, Japan. This product contains bacterial celluloseproduced during the fermentation process. A method for producingbacterial cellulose can also be found in JP10-95803.

Isolating, Purifying and Microfibrillating Cellulose

An example of preparing MFC from pulp of soft wood is known fromFranklin W. et al; Microfibrillated Cellulose: Morphology andAccessibility; Journal of applied Polymer Science; 1983; Applied Polymersymposium 37; pp. 797-813; John Wiley & Sons, Inc. The preparationmethod is described on page 798 in the section “Preparation ofmicrofibrillated cellulose” hereby incorporated by reference, and isfurther described on page 803 under “Discussion” in the section“Preparation of microfibrillated cellulose” also incorporated herein byreference. Also in EP 726 356 is a method for obtaining MFC described.

However, when isolating, purifying and microfibrillating celluloseuseful for preparing a film which is suitable for substituting and/ormimicking a cellulose containing textile in a screening process it ispreferred to employ methods and sources for which it is possible toobtain a long cellulose fibril structure, i.e. by avoiding breakage ofthe glucose chains constituting the cellulose and substantiallypreserving the original amounts of glucose units in the glucose chains.The method should deploy force to the cellulose fibres preferably onlyto expose the fibrils and microfibrils. The term substantially in thiscontext means that the DP (degree of polymerisation) of the glucosechains should be lowered by no more than 500 glucose units from theoriginal chain, preferably by no more than 350, more preferably by nomore than 250 and most preferably by no more than 150 glucose units bythe microfibrillation process.

A preferred cellulose is bacterial cellulose, which makes an excellentcellulose starting material for microfibrillation. Methods for obtainingcellulose from bacteria are known and described, such as from strains ofAcetobacter described in U.S. Pat. No. 4,863,565 examples VI and VII,incorporated herein by reference.

Microfibrillation of a bacterial cellulose, such as from the product“nata de coco” may be achieved by washing the cellulose in plenty ofwater to remove water soluble impurities, treating the washed cellulosewith an alkaline solution, such a NaOH and neutralising and blending thealkali treated cellulose to obtain a suspension of MFC.

Cellulose Films

The invention relates to a cellulose film comprising MFC. The film ofthe invention comprises preferably at least 50% w/w MFC, more preferablyat least 75% w/w, more preferably at least 95% w/w and most preferablythe film consist of substantially pure MFC. By using the termsubstantially it meant that small amounts of impurities originating fromthe source or from the purification or microfibrillation process mayremain in the film, but no substances has intently or deliberately beenadded to the film. A preferred film comprises MFC substantially having anative Cellulose I structure, meaning that a major portion of thecellulose have a Cellulose I structure. Another preferred film comprisesMFC substantially having a Cellulose II structure, while furtherpreferred films comprise cellulose substantially having Cellulose III orCellulose IV structures. Modification of cellulose structure is known tothe art. The conversion of Cellulose I to Cellulose III is for exampledescribed in Chanzy et al.; Structural changes of cellulose crystalsduring the reversible transformation cellulose I to cellulose III;Valonia. Holzforschung; 40; suppl. 25-30. Interactions between MFC ande.g. different endoglucanase enzymes was found to be highly dependent onthe cellulose structure.

The film have a preferred dry average thickness of about 10 μm to about100 μm, more preferably about 20 μm to about 70 μm and most preferablyabout 30 μm to about 60 μm.

Modified Cellulose Films

Because the cellulose film comprises MFC the accessibility of thecellulose is enhanced. Accordingly the cellulose in the film may bereacted and/or attached and/or blended/mixed with one or more compoundsor substances before or after formation of the film. Accordingly, in apreferred embodiment the cellulose film further comprises a compound orsubstance which before or after formation of the film has been reactedor attached or mixed with the MFC. Preferably the substance or compoundis reacted and/or attached onto the surface of the film after formationof the film. The compound or substance may be attached to the MFC bycovalent bonds or by ionic bonds or by hydrogen bonds such as byhydrophobic interaction between the compound or substance and the MFC orit may be mixed with the MFC before formation of the film, so that thesubstance or compound is embedded in a MFC matrix. Preferred compoundswhich may be attached to the MFC are compounds which possess optical orradioactive properties (often called markers or label agents) or whichupon release or attachments to the film gains optical properties or mayreact with optically detective indicators. Compounds which possessoptical properties or gain such properties may be reflectants orabsorbants, such as particles of pigments reflecting or absorbingmulti-wavelength light or more preferably dyes such as fluorescent dyesor light absorbing dyes, which emits or absorbs light at discretewavelengths. Examples of reflectants are indigo, opaque agents, carbonblack and/or titandioxide pigments. The dyes are typically conjugatedorganic molecules in which the conjugated system preferably is changedand the molecule either gain or loose fluorescence or absorbingproperties when reacted to or released from the film. However dyes forwhich the conjugated system does not change may also be used.Fluorescent dyes, such as DTAF, fluorescein,Fluorescein-isothiocyanate—Isomer I, or fluorescein-5-thiosemicarbazideare preferred.

Among the dyes suitable for labelling cellulose, derivatives of cyanurchloride are preferred because it has been found possible to react themto cellulose. In a method for labelling cellulose with derivatives ofcyanur chloride it has also been found the pH in the reaction medium iscrucial for obtaining satisfactory labelling. Accordingly we havedeveloped a method for labelling cellulose comprising reacting aderivative of cyanur chloride onto the cellulose at a pH between 9-10.

Other for attaching dyes to polysaccharides are known to the art and maybe found e.g. in WO 99/45143 incorporated herein by reference.

Radioactive compounds encompasses all compounds comprising radioactiveisotopes such as S³⁵, p³², H³ and/or I¹²⁵.

In a preferred embodiment the compound is a non-cellulose substrate fora non-cellulytic enzyme or a non MFC substrate, preferably comprising amoiety which possesses optical or radioactive properties as described,supra. The non-cellulytic enzyme substrate is preferably selected fromamino acids, peptides, proteins, carbohydrate polymers, oligomers ormonomers, fatty acids, fatty acid esters, fatty acid ester alcohols andtriglycerides. Accordingly the substrate may a polysaccharide such asstarch and/or a protein and/or a lipid.

Among dyes suitable for labelling acid groups, optically activederivatives of semithiocarbazid are preferred.

Among dyes suitable for labelling amine groups, optically activederivatives of isothioncyanate are preferred.

Also combinations of substrates are encompassed by the invention.Accordingly one useful combination is the combination of a cellulosefilm labelled with one dye mixed with a substantially amorphouscellulose such as CMC or PASC labelled with another dye. The termsubstantially in the context means that a major portion of the celluloseis in an amorphous form. When contacting such a film with an unknowncellulytic enzyme it may be identified if the enzyme mainly reacts withthe amorphous cellulose or the crystalline MFC by detecting which dye isreleased from the film.

The compound may also be a staining substance, i.e. the cellulose filmmay be stained with a substance, preferably containing a protein or alipid, fat or fatty acid or a polysaccharide or a naturally occurringcolorant or combinations thereof. As examples the stain may be made oftomato ketchup, grass, coffee, tea or animal lard.

Preparation of Cellulose Films

The invention also relates to a method for preparing a cellulose filmcomprising MFC comprising preparing a suspension of MFC andsedimentation of the MFC as a film onto a surface.

The surface may be any surface which is substantially impermeable to theMFC, i.e. the surface is impermeable to a major part of the MFC. Thesurface may be of any suitable material such as stainless steel alloys,plastics/synthetic polymers, rubber, board, glass, wood, paper, textile,concrete, rock, marble, gypsum and ceramic materials which optionallymay be coated, e.g. with paint, enamel, polymers and the like. Thesurface may however also be of biological origin such as mucousmembranes, skin, teeth, hair, nails etc.

In a preferred embodiment the film is prepared by preparing a suspensionof MFC in a container and sedimenting the MFC on at least one innersurface of the container, preferably the bottom surface of thecontainer. The bottom surface of the container is preferably made of asynthetic polymer such as a plastic, and may optionally be translucent.Accordingly in a most preferred embodiment the container is a well in amicrotiter plate, and preferably the microtiter plate contains 96 wellor more such as 384 well or 1536. Accordingly the container preferablyhave a volume of less than 10 ml, more preferably less than 1 ml, morepreferably less than 500 μl, more preferably less than 300 μl, morepreferably less than 50 μl and most preferably less than 15 μl. Byemploying such small containers film having a very small diameter may beprepared which is useful in a screening process. In order to sedimentMFC, substantially having a cellulose I structure, on a surface from asuspension the concentration of MFC in an aqueous suspension should beless than 10 mg/ml suspension, preferably less than 2 mg/ml, morepreferably less than 1 mg/ml and most preferably less than 0.7 mg/ml.For other cellulose structures these concentration may be higher, suchas multiplied by two. The film should preferably stick or adhere to thesurface and accordingly when preparing a film in container of dimensionscorresponding to a well of a 96 well microtiter plate the total amountof MFC sedimented and dried on the bottom surface should not exceed 250μg, preferably not exceed 200 μg and most preferably not exceed 150 μg.In such a container the best films are obtained by using about a 100 μlsuspension with a concentration of MFC of 1 mg/ml or less. An importantfeature of the invention is that cellulose films of the invention mayeasily be reproducibly prepared in a vast number of identical containerssuch as wells in a microtiter plate.

Applications of Cellulose Films

The invention also relates to the use of a cellulose film of theinvention for screening of actives, preferably biologically activecompounds, such as enzymes. Because the film can be preparedreproducibly and used in a vast number very small containers and mimicscellulose containing textile or fabric it is very useful for detectingactives, such as enzymes, which interacts with the cellulose orcompounds or substances attached to the cellulose.

Screening/identifying Actives

Most often screening for an active of interest requires contacting theactive with a substance which will undergo a detectable change uponreaction and/or interaction with the active. For actives such asenzymes, the skilled person will usually have a range of such substancesto choose from, but there is a desire to choose substances whichresembles substances with which the enzyme will react in an intendedreal life industrial application. For enzymes, choosing a real typesubstrate to which an interesting enzyme has a high specificity in thescreening process one advantage is that new enzymes found in thescreening process also are very likely to work well in the intendedindustrial application. Choosing e.g. a low molecular syntheticsubstrate of low specificity instead, however, may generate a largenumber of false positive hits in a screening, i.e. enzymes may be foundwhich reacts well with the synthetic substrate, but will perform poorlyon the real substrate in the intended industrial application. Byemploying the cellulose film of the invention a real life application ismimicked and enzymes found by a screening method employing the cellulosefilm of the invention are likely to interact with real textile cellulosein a desired way.

Accordingly the invention provides a method for screening for an active,preferably a biological compound comprising contacting, preferably in anaqueous medium, a sample containing the active with a cellulose filmcomprising MFC and detecting an interaction between the cellulose filmand the active.

In a preferred embodiment the method comprises the steps of:

(a) depositing a cellulose film of the invention on at least one innersurface of a container, preferably the bottom surface of a containerhaving a volume of less than 10 ml,

(b) adding the active dissolved or dispersed in a, preferably aqueous,liquid to the film,

(c) incubating the film with the active and

(d) monitoring the interaction between the biological compound and thecellulose film, preferably by measuring a compound which have beenreleased from the film by the interaction.

The released compound may in accordance with the invention be a dye,preferably fluorescent, or a radioactive compound or it may preferablybe a product of a substrate labelled with a dye or a radioactivecompounds which have reacted with the biological compound.

The active is preferably a selected from biological compound such as anenzyme and organic and inorganic detersive compounds. Relevant detersivecompounds may be enzyme stabilizers, inhibitors, enhancers, co-factors,builders, builder systems, bleach systems, bleach activators,metal-containing bleach catalyst, optical brighteners, nonionic-,anionic-, cationic-, zwitterionic and amphoteric surfactants, fabricsoftening agents, softening clays, clay flocculants, dye-transferinhibiting agents, polymeric soil release agents, clay soil removalagents, anti-soil redeposition agents, polymeric dispersing systems,chelating agents, alkoxylated polycarboxylates, carrier systems, dyesand pigments, fabric care agents, color care agents and like.

A preferred active is an enzyme. The enzyme may be a cellulose degradingor synthesising enzyme which interacts directly with the cellulose inthe film and the presence of such enzymes may be detected by measuringthe release of glucose oligo- or monomers from the film or theconsumption of glucose oligo- or monomers from the medium in which theinteraction occurs. Methods for detecting glucose oligo- or monomers areknown to the art, e.g. from Kidby D. K. and Davidson d. J.; A convenientferricyanide estimation of reducing sugar in the nanomole range;Analytical Biochemistry; 1973; 55; pp. 321-325. Such enzymes may beendoglucanases or cellulases such as those belonging to the groupendo-1,4-beta-glucanase (EC 3.2.1.4) or endo-1,3(4)-β-glucanases (EC3.2.1.6).

The enzyme classification employed is in accordance with Recommendations(1992) of the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology, Academic Press, Inc., 1992.

The enzyme may equally preferred be a non-cellulose degrading enzymewhich interacts with a substrate attached to the film or which isomerizecellulose. It is to be understood that enzyme variants (produced, forexample, by recombinant techniques) are included within the meaning ofthe term “enzyme”.

Accordingly the types of enzymes which may appropriately be screenedinclude oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-),hydrolases (EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-)and ligases (EC 6.-.-.-).

Preferred oxidoreductases in the context of the invention areperoxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC1.1.3.4)].

Preferred transferases are transferases in any of the followingsub-classes:

a) Transferases transferring one-carbon groups (EC 2.1);

b) transferases transferring aldehyde or ketone residues (EC 2.2);acyltransferases (EC 2.3);

c) glycosyltransferases (EC 2.4);

d) transferases transferring alkyl or aryl groups, other that methylgroups (EC 2.5); and

e) transferases transferring nitrogeneous groups (EC 2.6).

A most preferred type of transferase in the context of the invention isa transglutaminase (protein-glutamine γ-glutamyltransferase; EC2.3.2.13).

Preferred hydrolases in the context of the invention are: Carboxylicester hydrolases (EC 3.1.1.-) such as lipases (EC 3.1.1.3); phytases (EC3.1.3.-), e.g. 3-phytases (EC 3.1.3.8) and 6-phytases (EC 3.1.3.26);glycosidases (EC 3.2, which fall within a group denoted herein as“carbohydrases”), such as α-amylases (EC 3.2.1.1); peptidases (EC 3.4,also known as proteases); and other carbonyl hydrolases].

In the present context, the term “carbohydrase” is used to denote notonly enzymes capable of breaking down non-cellulose carbohydrate chains(e.g. starches) of especially five- and six-membered ring structures(i.e. glycosidases, EC 3.2), but also enzymes capable of isomerizingcarbohydrates, e.g. six-membered ring structures such as D-glucose tofive-membered ring structures such as D-fructose.

Carbohydrases of relevance include the following (EC numbers inparentheses):

α-amylases (EC 3.2.1.1), β-amylases (EC 3.2.1.2), glucan1,4-α-glucosidases (EC 3.2.1.3), , endo-1,4-β-xylanases (EC 3.2.1.8),dextranases (EC 3.2.1.11), chitinases (EC 3.2.1.14), polygalacturonases(EC 3.2.1.15), lysozymes (EC 3.2.1.17), β-glucosidases (EC 3.2.1.21),α-galactosidases (EC 3.2.1.22), β-galactosidases (EC 3.2.1.23),amylo-1,6-glucosidases (EC 3.2.1.33), xylan 1,4-β-xylosidases (EC3.2.1.37), glucan endo-1,3-β-D-glucosidases (EC 3.2.1.39), α-dextrinendo-1,6-α-glucosidases (EC3.2.1.41), sucrose α-glucosidases (EC3.2.1.48), glucan endo-1,3-α-glucosidases (EC 3.2.1.59), glucan1,4-β-glucosidases (EC 3.2.1.74), glucan endo-1,6-β-glucosidases (EC3.2.1.75), arabinan endo-1,5-α-L-arabinosidases (EC 3.2.1.99), lactases(EC 3.2.1.108), chitosanases (EC 3.2.1.132) and xylose isomerases (EC5.3.1.5).

The invention also relates to a biological compound identified themethod of the invention.

The sample to be screened may contain the active in a crude or apurified form or it may in case the active is a biological compoundcontain cells or in vitro coupled transcription and translation systemwhich produce or have produced the biological compound. The cells may bebacterial cells, archaeal cells and/or eucaryotic cells.

In a preferred embodiment the active is an enzyme in a detergentcomposition. It is known to the art that enzyme properties such asactivity and stability may be altered or inactivated by the presence ofdetergents. Accordingly it is desired to screen for an enzyme in thepresence of a detergent because enzymes which are more effective indetergent compositions may be identified. Accordingly the screeningmethod of the invention may advantageously replace such screeningmethods known to the art e.g. as described in WO 99/34011.

Screening For Nucleic Acid Sequences

As biological compounds, which can be screened and detected by themethod of the invention may be expressed by a cell or an in vitro systemencoded by nucleic acid sequences comprised in the cell or in vitrosystem, also nucleic acid sequences encoding a biological compound maybe screened and identified and isolated.

Accordingly the invention also provides a method for screening a nucleicacid sequence encoding a biological compound, wherein the methodcomprises:

(a) expressing a nucleic acid sequence in an expression system, so as toproduce a biological compound,

(b) contacting the biological compound with a cellulose film preferablycomprising MFC,

c) measuring an interaction between the biological compound and thecellulose film and

d) selecting expression systems for which a detectable interactionoccurred and recovering the nucleic acid sequence.

Nucleic Acid Sequence Sources

The nucleic acid sequence originates from a source. In a preferredembodiment of the invention the source of the nucleic acid the bescreened is a cell, e.g. a prokaryotic cell, an archaeal cell or aneucaryotic cell. The cell may further have been modified by geneticengineering. A preferred bacterial cell is of the genus Bacillus, e.g.B. licheniformis, while a preferred eucaryotic cell is a mammal cell,e.g. a human cell, a plant cell, e.g. Arabidopsis thaliana or a fungus,e.g. Meribipilus gigantus.

In another preferred embodiment the nucleic acid source is a mixedpopulation of cells. The DNA or RNA of the cells may further beextracted, as described vide infra, directly from any biotic or abioticsample, e.g. a soil sample, a water sample, or a rumen sample. Alsopreferred nucleic acid sources are cells of extremeophile prokaryotics,such as thermophiles.

The nucleic acid source may also be cells which have been subjected toclassical mutagenesis, e.g. by UV irradiation of the cells or treatmentof cells with chemical mutagens as described by Eisenstadt E., CarltonB. C. and Brown B. J., Gene mutation, Methods for general and molecularbacteriology, pp. 297-316, Eds: Gerhardt P., Murray R. G. E., Wood W. A.and Krieg N. R., ASM, 1994.

Further the nucleic acid source may be a population of cells geneticallymodified by in vivo gene shuffling as described in WO 97/07205.

In a further preferred embodiment the nucleic acid source is in vitromade preparations of sequences of DNA, RNA, cDNA or artificial genesobtainable by e.g. gene shuffling (e.g. described by Stemmer, Nature,370, pp. 389-391, 1994 or Stemmer, Proc. Natl. Acad. Sci. USA, 91, pp.10747-10751, 1994 or Wo 95/17413), random mutagenesis (e.g. described byEisenstadt E., Carlton B. C. and Brown B. J., Gene mutation, Methods forgeneral and molecular bacteriology, pp. 297-316, Eds: Gerhardt P.,Murray R. G. E., Wood W. A. and Krieg N. R., ASM, 1994) or constructedby use of PCR techniques (e.g. described by Poulsen L. K., Refn A.,Molin S. and Andersson P., Topographic analysis of the toxic Gef proteinfrom Escherichia coli, Molecular Microbiology, 5(7), pp.1627-1637, 1991)

Expression Systems

In the method of the invention nucleic acid sequences to be screened areexpressed in an expression system. The expression system is a systemenabling transcription of a nucleic acid sequence and translation intothe synthesis of the corresponding biological compound. The expressionsystem may be cellular or an in vitro system. A description of in vitrocoupled transcription and translation may be found in Ohuchi, S. et al.;In vitro method for generation of protein libraries using PCRamplification of a single DNA molecule and coupledtranscription/translation; Nucleic Acid research, 1998, vol. 26. No. 19,pp. 4339-4346 or Ellman J., Mendel D., Anthony-Cahill S. J., Noren C. J.and Schultz P. G., Methods in Enzymol.1991; vol. 202; pp. 301-337,enabling expression of a nucleic acid sequence, e.g. a gene libraryderived from a nucleic acid source. In the case of a cellular expressionsystem, the cell may be the nucleic acid sequence source itself, e.g. awild type cell isolated from nature, or it may be a cell from apopulation of transformed host cells or clones thereof comprising a genelibrary prepared from a nucleic acid source according to methods knownto the art (e.g. described vide infra).

Host Cells

The host cell according to the definition may be any cell able ofhosting and expressing a nucleic acid fragment from a gene library.

A preferred host cell does not in itself contain or express nucleic acidsequences encoding for biological compounds (i.e. untransformed hostcells are unable of significantly expressing the biological compound),which will interfere with the screening method. This cell characteristicmay either be a natural feature of the cell or it may be obtained bydeletion of such sequences as described e.g. in Christiansen L. C.,Schou S., Nygaard P. and Saxild H. H., Xanthine metabolism in Bacillussubtilis: Characterization of the xpt-pbux operon and evidence forpurine and nitrogen controlled expression of genes involved in xanthinesalvage and catabolism, Journal of Bacteriology, 179(8), pp 2540-2550,1997 or Stoss O., Mogk A. and Schumann W., Integrative vector forconstructing single copy translational fusions between regulatoryregions of Bacillus subtilis and the bgaB reporter gene encoding a heatstable beta-galactosidase, FEMS Microbiology Letters, 150(1), pp 49-54,1997.

In another preferred embodiment of the invention the host cell is abacterial cell or an eucaryotic cell. Further the bacterial cell ispreferably a ElectroMAX DH10B (GibcoBRL/Life technologies, UK)cell or ofthe genus E. coli, e.g. SJ2 E. coli of Diderichsen, B., Wedsted, U.,Hedegaard, L., Jensen, B. R., Sjoholm, C., “Cloning of aldB, whichencodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillusbrevis”, J. Bacteriol., 172, pp 4315-4321, 1990. Other preferred hostcells may be strains of Bacillus, such as Bacillus subtilis or Bacillussp. A preferred eucaryotic cell is preferably a yeast, e.g. S.cerevisae.

Preparation of Gene Libraries

Preparation of a gene library can be achieved by use of known methods.

Procedures for extracting DNA from a cellular nucleic acid source andpreparing a gene library are described in e.g. Pitcher, D. G., Saunders,N. A., Owen, R. J., “Rapid extraction of bacterial genomic DNA withguanidium thiocyanate”, Lett. Appl. Microbiol., 8, pp 151-156, 1989 orDretzen, G., Bellard, M., Sassone-Corsi, P., Chambon, P., “A reliablemethod for the recovery of DNA fragments from agarose and acryla-midegels”, Anal. Biochem., 112, pp 295-298, 1981 or WO 94/19454 orDiderichsen et al., supra.

Procedures for preparing a gene library from an in vitro made syntheticnucleic acid source can be found in (e.g. described by Stemmer, supra orWO 95/17413).

Insertion of Gene Libraries Into Host Cells

Procedures for transformation of a host cell by insertion of a plasmidcomprising a DNA or cDNA fragment from a gene library is well known tothe art, e.g. Sambrook et al., “Molecular cloning: A laboratory manual”,Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; 1989 or Ausubel, F.M. et al. (eds.) Current protocols in Molecular Biology, John Wiley andSons, 1995 and Harwood, C. R., and Cutting, S. M. (eds.), “MolecularBiological Methods for Bacillus”, John Wiley and Sons, 1990.

In a preferred embodiment of the invention the plasmid to be insertedinto a host cell also contains a nucleic acid sequence (denoted as anantibiotic marker), which may enable resistance of a transformant to anantibacterial or antifungal agent e.g. an antibiotic. Resistance tochloramphenicol, tetracycline, kanamycin, ampicillin, erythromycin orzeocin is preferred.

In a further preferred embodiment of the invention the pSJ1678 μlasmidDNA of WO 94/19454 and Diderichsen et al. (1990), supra, which enablesresistance to chloramphenicol, may be used for transforming a SJ2 E.coli host cell. Alternatively the plasmid pZErO-2 (Invitrogen, CA, USA)may be used).

Screening Procedures

In the case of an in vitro expression system as a specific embodiment ofthe invention the screening procedure preferably comprises:

a) preparing a gene library from a nucleic acid source,

b) separating the gene fragments of the library into separatecontainers.

c) amplifying the separated gene fragments,

d) performing in vitro coupled transcription/translation of theamplified gene fragments so as to express a biological compound.

e) contacting the biological compound in each separate container orsubsamples thereof with a cellulose film of the invention,

f) incubating the biological compound with the cellulose film,

g) detecting an interaction between the cellulose film and thebiological compound,

h) recovering gene fragments in containers in which an interaction hasoccurred

Steps a-h may suitably be achieved by use of commercially availablestandard equipment such as pipettes or automated pipette equipment,flasks, microtiter plates, shakers, thermostated incubators etc.

An interaction between a biological compound and the cellulose filmoccurs only in containers containing and expressing a gene fragment ornucleic acid sequence encoding for a biological compound.

The separation of gene fragments of the library may be achieved bydiluting the library to a degree which enables sampling of aliquotscontaining a gene fragment, preferably an average of one gene fragmentper sample and then transferring the samples to separate containers,e.g. microtiter wells. The amplification of the separated gene fragmentsmay be achieved by conventional PCR techniques as well as the in vitrocoupled transcription/translation of the amplified gene fragments (seeOhuchi et al. (1998), supra, page 4340 or Ellman et al. (1991), supra,which is hereby incorporated by reference.

In the case of a cellular expression system as a specific embodiment ofthe invention the screening procedure preferably comprises:

a) pre-propagating and dilution of cells comprising the nucleic acidsequence,

b) separating the cells into separate containers,

c) propagating separated cells to increase the number of clones of eachcell in each separate container,

d) contacting the cells in each separate container with a cellulose filmof the invention,

e) incubating the biological compound with the cellulose film,

f) detecting an interaction between the cellulose film and thebiological compound and

g) recovering gene fragments in containers in which an interaction hasoccurred

Steps a-g may suitably be achieved by use of commercially availablestandard equipment such as pipettes or automated pipette equipment,flasks, microtiter plates, shakers, thermostated incubators etc.

Pre-propagation and dilution of the cells may in one embodiment of theinvention be designed to obtain a concentration of cells per volumesuitable for sampling aliquots containing an optimal number of cells tobe separated. A suitable average number cells per aliquot may be 0,3-10,preferably 0,3-5, e.g. 0, 3-1.

Pre-propagation of the cells in a preferably aqueous medium preferablyprovides a 2-5 times increase in the number of cells clones. Suitableincubation temperatures may be within the range of 10-60° C., preferable30-50° C., e.g. 37° C., while pH may be kept between 4-10, preferably6-8. The incubation period should be adjusted, preferably 15-60 minutes,e.g. 40 minutes, so as to meet the requirements of the desiredtransformant and clone concentration.

In the case of the expression system being a culture of host cellswherein transformants comprises a gene library derived from a nucleicacid source, pre-propagation may also be performed to secure expressionof an antibiotic marker which may be comprised in the inserted plasmidof transformed host cells enabling resistance to an antibiotic in themedium. Pre-propagation of the host culture may accordingly be achievedby incubation at conditions favorable for expression of the chosen typeof antibiotic marker as well as securing viability of the transformant.

Dilution may be performed by addition of a medium, to ensure that allcells and clones thereof resides in the diluted solution.

The cells may be separated by transferring aliquots of the plannedspecific volume to separate containers, e.g. wells in commercialmicrotiter plates.

The separated cells are propagated to increase the number of clones ineach container, preferably to a range between 10⁷⁻10⁸ clones/ml. Ifmicrotiter plates are used these plates may be denoted “master plates”.For screening a bacterial gene library 50-100 master plates with each 96wells may typically be employed. One advantage of having a master plateis the possibility of keeping viable samples of the cells to bescreened, so that even if the subsequent screening conditions results indeath of the screened cells, it is possible to track back a screeningresult to a viable sample of a screened cell.

In a further preferred embodiment suitable incubation temperatures maybe in the range of 10-60° C., preferable 30-50° C., e.g. 37° C., whilethe pH may be kept between 4-10, preferably 6-8. The incubation mediumshould meet the nutritional requirements of the cells and clones toensure sufficient growth.

Also in the case of the expression system being a culture of host cellswherein transformants comprises a gene library derived from a nucleicacid source and an antibiotic marker a medium may suitable be chosenenabling killing or suppressing non-transformed host cells.

Incubation times should be adjusted so as to ensure growth yielding asufficient number of cells/clones suitable for sampling aliquotscontaining a suitable number of clones for screening leaving a number ofviable clones in the master plate. A preferred propagation period may be40-90 hours, e.g. 48 hours, depending on the type of microbial host celland the propagation conditions.

In the case of the expression system being a culture of host cellscomprising a gene library in which an antibiotic marker is comprised intransformed host cells the pre-propagation, dilution and/or propagation,in a preferred embodiment of the invention, are performed in a mediumcapable of selectively killing or suppressing growth of non-transformedhost cells. This may preferably be achieved by adding e.g. an antibioticto the culture medium towards which the transformants or clones thereofare resistant, in an amount effective on non-transformed host cells.

Aliquots of the cells are transferred, e.g. by pipette, to a microtiterplate containing the cellulose film of the invention. The contactbetween the biological compound and the cellulose film occur via theextracellular medium. In case the biological compound is confined withinthe interior of the cell, the cell may be lysed or its integrityotherwise disrupted in order to release the biological compound to themedium.

The incubation may be performed at conditions, which favor the reactionbetween the biological compound and cellulose film. In a preferredembodiment the reaction between the fluorescent substance(s) and thebiological compound is optimized with respect to pH and temperature. Theincubation may also be performed at extreme conditions (such as very lowtemperatures (e.g. below 30° C. or below 20° C.) or high temperatures(e.g. above 60° C. or above 70° C.), low pH (e.g. below 5 or below 4) orhigh pH (e.g. above 9 or above 10), low or high ionic strength, presenceof hostile chemicals such as detergents) causing death of the cells,depending on which biological compound is to be detected. If forinstance the biological compound to be found is a thermostabile compoundthe incubation may be performed at high temperatures, conditionsproviding that only biological compounds, which remain active at hightemperatures will react with a cellulose film of the invention.

Nucleic acid sequences in a cellular nucleic acid source or a genelibrary derived from a nucleic acid source and expressed either in an invitro expression system or by transformation into a host cell expressionsystem may thus be screened for nucleic acid sequences encoding forbiological compounds which reacts with the cellulose film of theinvention.

Also the invention relates to nucleic acid sequences encoding abiological compound found by employing the screening method of theinvention and to a method for producing a biological compound comprisingthe steps of

a) Identifying in a population of cells or in vitro expression systems,cells or systems which expresses a biological compound by contactingcells of the population with a cellulose film of the invention,

b) selecting cells or systems producing the biological compound

c) identifying a nucleic acid sequence encoding the biological compound

d) Cultivating a cell comprising a nucleic acid sequence encoding thebiological compound so as to produce the biological compound and

e) recovering the biological compound.

The invention is illustrated by the following examples, which is not inany way intended to be limiting to the scope of the invention.

EXAMPLES Example 1

Bacterial cellulose microfibrils in an impure form was obtained from theJapanese food stuff “nata de coco” (Fujico Company—Kobe Japan). Thecellulose in 350 g of this product were purified by suspension of theproduct in about 4 L of tap water. This water was replaced by freshwater twice a day for 4 days. Then, 1% NaOH (w/v) was used instead ofwater and the product was re-suspended in the alkali solution twice adays for 4 days. Neutralisation was achieved by rinsing the purifiedcellulose with distilled water until the pH at the surface of theproduct was neutral. The cellulose was microfibrillated and a suspensionof individual bacterial cellulose microfibrils was obtained byhomogenisation of the purified cellulose microfibrils in a Waringblender for 30 min. The cellulose microfibrils were further purified bydialysing this suspension through a pore membrane against distilledwater and the isolated and purified cellulose microfibrils were storedin suspension at 4° C. Diluted suspensions of bacterial cellulose weredeposited on carbon coated electron microscope grids and the structureof the isolated cellulose microfibrils was recorded by a Phillips CM 200Cryo transmission electron microscope (T.E.M.). The results showed thatthe individual bacterial cellulose microfibrils have a ribbon-likemorphology. The width of these microfibrils is of about 100 nm and theirthickness estimated from the twist of the microfibrils is in average of5 nm.

Example 2

The preparation method of example 1 required more than a week to obtainthe isolated and purified suspensions of bacterial cellulosemicrofibrils. Accordingly an alternative preparation method wasdeveloped which took only two days without modifying the properties ofthe bacterial cellulose microfibrils. The cellulose in 350 g of “nata decoco” were rinsed extensively with tap water in order to remove theexcess impurities such as of sweeteners and flavours. The partiallypurified cellulose was separated and were then re-suspended andmicrofibrillated by homogenisation in water using a Waring blender for10 minutes. This cellulose microfibril suspension was separated andre-suspended twice in 1% NaOH by centrifugation and kept in the alkalisolution under stirring overnight at room temperature. The purifiedsuspension of cellulose microfibrils was neutralised by at least threecentrifugations and re-dispersions of the specimen in water. Theresulting purified and isolated cellulose microfibrils was treated at70° C. for 1-2 hours by a bleaching solution consisting of 1 volume partof 1.7% aqueous NaClO₂ and 1 volume part of acetate buffer (pH 4.9)completed with 3 volume parts of distilled water. Finally, the bacterialcellulose microfibrils were washed from the bleaching solution byseveral centrifugations with distilled water. The purified and isolatedcellulose microfibrils was homogenised in a distilled water suspensionwith a Waring blender for 20 min and stored at 4° C.

Example 3

The bacterial MFC having Cellulose I structure obtained as described inexample 1 and 2, was converted into Cellulose III_(I) according to theprocedure in Chanzy et al.; Structural changes of cellulose crystalsduring the reversible transformation cellulose I to cellulose III;Valonia. Holzforschung; 40; suppl. 25-30. Microfibrils of bacterialcellulose obtained in example 1 was suspended in pure methanol and wastransferred into anhydrous ethylene-diamine after centrifugation. Themixture was kept overnight at room temperature in ethylene-diaminebefore being re-suspended for few hours in pure methanol. The wholetreatment was repeated six time until the complete conversion ofcellulose I into the cellulose III_(I) was observed. The transformationwas recorded by X-ray diffractometry and Fourier transform Infra-redspectroscopy. Dried samples of cellulose I microfibrils and celluloseIII_(I) microfibrils were analysed by X-ray with a Warhus flat filmcamera mounted on Philips PW1720 X-ray generator emitted Ni filteredCuKa radiation operated at 30 kV and 20 mA. When the conversion ofnative cellulose into cellulose IIIwas not completed intermediatepatterns were observed.

For Fourier Transform-Infrared (FT-IR) spectroscopy, drops of cellulosesuspensions were dried in a polyethylene cap at 50° C. The films werecarefully collected and mounted on the specimen holder before beinganalysed with a Fourier transform Infra-red Perkin Elmer 1720Xspectrometer. The spectra were recorded in the transmission mode with aresolution of 4 cm⁻¹ in the range of 4600 to 400 cm⁻¹. The conversion ofnative cellulose into cellulose III_(I) leads to remarkablemodifications of the spectra. The most noticeable transformations areextinction of characteristic peaks of cellulose I at 710 cm⁻¹ and 750cm⁻¹ and the appearance of an intense sharp peak at 3480 cm⁻¹characteristic of cellulose III_(I).

Example 4

Cotton bolls grown in a green house at the Texas Tech University(Lubbock) in 1989 and kept at 4° C. in water with sodium azide were usedas starting material. The seeds coated with cellulose fibres wereremoved from the bolls under water. Still under water, cellulose wasseparated from the seeds with tweezers and were cut into small fragmentswith a pair of scissors. The long cellulose fibres were shortened andhomogenised with a Waring blender until the large cellulose aggregateshad disappeared. Then, the cellulose specimen was microfibrillated inwater twice in 1 hour with an APV Gaulin homogeniser. The MFC wasre-suspended in 1 N NaOH overnight under stirring. The purification ofcellulose microfibrils was followed by a treatment with a bleachingsolution as in example 2 for one hour at 70° C. After an extensivewashing of the cellulose microfibrils with distilled water bycentrifugation, the isolated cotton microfibrils in suspension wasstored at 4° C. As in example 1 the structure of the cellulosemicrofibrils were examined by transmission electron microscopy, whichshowed that the original cotton fibres were disrupted into microfibrilsand microfibril bundles. The mechanical treatments applied to the cottonfibres had induced the delamination of the cellulose into flat bundles100-500 nm long. Such bundles are composed of the tight association ofmicrofibrils, 5-10 nm in width, that have been partly individualisedduring the treatment. Individual microfibrils can be seen in thebackground of the image but more frequently at the surface of thebundles to which they remain associated.

Example 5

The native bacterial cellulose I microfibrils of example 1 and thoseconverted into cellulose III_(I) from example 3 tested as substrates forcellulases. Test enzymes were Humicola insolens complex enzymes, whichis a complex of enzymes recoverable from the supernatant when fermentingthe fungus Humicola insolens; and the endoglucanases V and VI describedin Schou C. et al.; Stereochemistry, specificity and kinetics of thehydrolysis of reduced celludextrins by nine cellulases; Eur. J.Biochem.; 1993; 217; pp.947-953; and Schulein et al.; “Humicolainsolens, alkaline cellulases”; in “Trichoderma reesei cellulases andother hydrolases”; (eds. Suominen P. et Reinikainen T.); Foundation forBiotechnical and Industrial Fermentation Research; Helsinki; vol. 8; pp109-116. The procedure was conducted as follows: 75 μl of enzymessolution (1 mg/ml) was mixed with aliquots of 600 μl of cellulose (100μ0g/100 μl) microfibrils suspended in 50 mM phosphate buffer at pH 6.5.The digestion was achieved at 37° C. without agitation.

The degradation kinetics of cellulose microfibrils were followed bymeasuring the amount of reducing sugars in the supernatant aftercentrifugation of the degradation mixture according to the ferricyanidemethod adapted from the Kidby and Davidson (1973), supra. 100 μl of theassays supernatant were treated in boiling water for 7 min. by 1 ml ofthe ferricyanide solution that consisted of the mixture of 300 mg ofpotassium hexacyanoferrate III, 28 g of hydrated sodium carbonate(NaCO₃, H₂O) and 1 ml of 5M NaOH completed to 1 L with distilled water.The absorbency of the solutions was measured at 420 nm, theconcentration of reducing sugars was calculated using standard curveobtained using glucose solutions of known concentration.

The results are shown in FIG. 1 in which the two curves depict thedigestion kinetics of cellulose I and III_(I) bacterial microfibrils bythe H. insolens complex. It appears in this illustration that the amountof solubilised reducing sugars produced at extended time is very similarfor both substrates. In contrast, the reactivity of the cellulosesubstrates was markedly different when they were incubated withendoglucanases V or VI as shown in FIGS. 2A and B. Indeed, in FIG. 2, itis observed that the extent of degradation was multiplied by a factor ofabout 9 and 5 for EG V and EG VI respectively when going from celluloseI to cellulose III_(I).

Example 6

The fluorescent dye 5-5([4,6-dichlorotriazin-2-YL]amino) fluorescein(DTAF) was attached or grafted on MFC I in a single step procedure. Thetriazino reactive group of DTAF was known to be quite reactive on thehydroxyl groups of polysaccharides. Consequently, the DTAF molecule wasa good candidate for preparing fluorescent cellulose.

One-set MFC grafted with DTAF was prepared by mixing 10 to 70 mg of DTAF(Sigma) with 10 ml of native bacterial cellulose (10 mg/ml) insuspension in 0.1 M NaOH. These mixtures were kept at room temperaturefor 24 hours under stirring. Then, the cellulose specimens were washedfree of unreacted DTAF by at least six centrifugations with distilledwater. A second set of derivatised cellulose was prepared as the firstset, but the amount of DTAF was in the range of 70 to 115 mg and theconcentration in 0.2 M NaOH.

Preliminary experiments revealed that is was not possible to estimateeasily and quickly the extent of cellulose labelling by spectroscopicmethod. Consequently, we incubated labelled cellulose microfibrils withcellulases (H. insolens complex, endoglucanases EGV or EGVI) assumingthat the release of the fluorescent probe in the supernatant ofcentrifuged assays should increase with the amount of DTAF grafted ontothe surface of cellulose.

Tests were performed by adding 20 μl of H. insolens complex (1 mg/ml) to600 μl of labelled cellulose (100 μg/100 μl) in 50 mM phosphate buffer.The mixtures were incubated for 4 hours at 37° C. without agitation. Inparallel, control experiments were conducted using water instead ofenzyme in order to visualised the non specific release of thefluorescent probe. Supernatants of the corresponding assays collectedafter centrifugation were diluted 4 times with distilled water. Thefluorescence of 200 μl of each specimens was recorded using distilledwater as control. For each assay, the relative intensity (R.I.) offluorescence release during the enzymatic digestion was deduced bysubtracting the fluorescence of the test containing the enzyme from itscorresponding control test. In FIG. 3, it is observed that when thechemical reaction was conducted in 0.1 M NaOH, the release of thefluorescent probe increases until the amount of DTAF used was of about40 mg/100mg of MFC. When higher concentration of DTAF was reacted withMFC, the intensity of fluorescence remained constant, suggesting thatthe level of grafting did not increase. For the set of graftingexperiments performed in 0.2 M NaOH, the amount of DTAF solubilisedafter the enzymatic treatment is higher than when the labelling was donein 0.1 N NaOH. Nevertheless, the release of the probe was constant anddid not increase with the amount of DTAF. Consequently, a single-stepgrafting experiments allowed to reach only a limited range ofderivatisation of the MFC.

Example 7

DTAF was attached or grafted on MFC I in a multi-step procedure. A firstseries of cellulose labelling assays was conducted by mixing 30 mg ofDTAF with 10 ml of native cellulose suspension (10 mg/ml) in 0.1M NaOH.The mixture was stirred for 24 hours at room temperature. Then, thespecimens were washed extensively by centrifugation with distilledwater. The above procedure was repeated several times (steps) andcellulose suspensions were stored at 4° C. A second series of assays wasperformed according to the same conditions excepted that DTAF was addedby steps of 60 mg and the alkali reaction medium was of 0.2M NaOH.

Tests of the enzymatic degradation of the labelled MFC was achieved asfor the single-step experiments: samples of 600 μl of grafted MFCsuspension (100 μg/100 μl ) in 50 mM phosphate buffer at pH 6.5 weremixed with 20 μl of the H. insolens complex (1 mg/ml), or 20 μl of EG VI(1 mg/ml) or 20 ml water as standard. Assays were conducted for 4 hoursat 37° C. without agitation. Supernatants of the respective centrifugedspecimens were analysed by the ferricyanide method to measure theconcentration of soluble reducing sugars and by spectrofluorometry toestimate the range of release of the fluorescent probes.

The results on incubating the first series of labelled cellulose with H.insolens complex and EG VI, shown in FIGS. 4 and 5 respectively, leadsto a strong release of the fluorescent probes which was diluted 8 timeto scale down the intensity in the range of the spectrofluorometersensibility. In FIG. 4, the increase of fluorescence with the numbersteps of labelling experiments indicates that the number of DTAFmolecule linked to the surface of the cellulose microfibrils increasedas well. Also, the decrease of the soluble reducing sugars producedduring enzymatic attack when the grafting increase can be easilyinterpreted as an inhibition of the enzyme by the grafted molecules thatcover the surface of cellulose.

The degradation of the labelled cellulose by EG VI is shown in FIG. 5.As for H. insolens complex, the fluorescence did increase with thenumber of labelling experiments. However, inhibition of the enzymebecause of the labelling does not seem to occur, the concentration ofthe reducing sugars solubilised being quite constant. But, it should benoticed that the amount of soluble reducing sugar is quite low and thatthe ferricyanide method detection may not allow to evidence easily veryfine variation in their concentration.

FIGS. 6 and 7 shows the results of degradation experiments on MFC thatwas grafted by a number of steps with 60 mg of DTAF in 0.2 N NaOH. Inboth cases, the action of the enzymes—H. insolens complex or EG VI—leadsto an increase of the fluorescence until a maximum was observed for MFCgrafted four times. For MFC grafted more than four times a decrease ofthe fluorescence is detected. In the case of H. insolens complex (FIG.6), the variation of fluorescence is clearly associated with a regulardecrease of the concentration of soluble reducing sugars that isconsistent with an inhibition of the enzymes by the linked DTAFmolecules onto cellulose microfibrils. This suggest that for the fourfirst steps of times of labelling, the increase in amount of fluorescentsugars solubilised is more important than the decrease of total amountof reducing sugars. When the inhibition of the enzyme becomes stronger,the amount of grafted sugars released is markedly reduced leading to adecrease of the fluorescence. The same interpretations of the resultsobtained with EG VI presented in FIG. 7 could be done except that theinhibition of the enzyme could not be evidenced clearly with theferricyanide method.

Example 8

DTAF was attached or grafted on MFC III_(I) in a multi-step procedure.The grafting of DTAF on MFC III_(I) was achieved according to the sameprocedure described in example 7 using 60 mg of DTAF in 0.2 N NaOH. Asfor the MFC I, the grafted MFC III_(I) was incubated with cellulases andthe results of the degradations using 16 times diluted samples are shownin FIGS. 8 (H. insolens ) and 9 (EG VI). The variation of fluorescencefollows the same behaviour as that of the grafted MFC I. However, themaximum release of the probe was obtained when the cellulose III_(I) wasreacted three times with 60 mg of DTAF (0.2 N NaOH) instead of fourtimes with MFC I. The inhibition of the H. insolens complex is alsoclearly visible by the regular decrease of reducing sugars produced whenthe level of grafting increase. Also, in the case of EG VI, it isobserved in FIG. 9, that the amount of reducing sugar produced decreaseswith amount of grafted DTAF.

As it was showed in example 5, MFC III_(I) is more reactive towardsendoglucanases than microfibril-lated cellulose I. This was evidenced byan increase of the total amount of reducing sugars solubilised. Also,the labelled MFC III_(I) is more reactive than the labelled MFC Ileading to an increase of the reducing sugars liberated in theincubation medium. Consequently, the ferricyanide method which did notseem sensitive enough according to our procedure with MFC I allowed toreveal the inhibition of EG VI by the grafted cellulose III_(I).

Example 9

Films of MFC I was prepared. Suspensions of various concentration of MFCin water in the range of 0.3 mg/ml to 2 mg/ml were maintained at roomtemperature without agitation to allow sedimentation of MFC. After fewhours, MFC I of the less concentrated suspensions (0.3 mg/ml-0.7 mg/ml)had sedimented. However, for the more concentrated suspensions (=1mg/ml), the cellulose did not sediment even after several days. In thecase of MFC III_(I), the sedimentation of cellulose was observed whenthe concentration of the suspensions was lower than 1.5 mg/ml.

Deposition of MFC on the bottom of 96 well microtiter plate (Nunc-immunoPlateMaxsorp™, Nunc) were achieved by drying at 37° C. various volumesof suspensions of MFC (50, 100 and 200 μl) of various concentration (0.1mg/ml to 2 mg/ml). It appeared rapidly that the films did not stick ontothe surface of the wells when the total amount of dried MFC was morethan 150 mg. Also, when the volume of suspensions was superior to 200ml, the cellulose adhered onto the wall of the well in a nonreproducible fashion. It was found that the best films were obtainedwith 100 μl of suspension having a concentration of about 1 mg/ml orbelow.

Example 10

Enzymatic degradation of unlabelled films was tested. Films of MFC Iwere obtained by drying 100 μl of cellulose I suspensions (1 mg/ml) at37° C. per well of a 96 well microtiter plates. The reproducibility ofthe films was tested towards their susceptibility to enzymaticdegradation: 200 μl of 50 mM phosphate buffer at pH 6.5 followed by 20μl of H. insolens complex (1 mg/ml) were added in each well and kept at37° C. At various incubation times, 8 samples of 100 μl were collectedand the amount of solubilised reducing sugars produced were measured bythe ferricyanide method. The average and the standard error werecalculated according to the following equations where n is the number ofsamples and x the amount of reducing sugars.${{Average} = {\overset{\_}{X} = \frac{\sum\limits^{1arrow n}x_{n}}{n}}}\quad$${{Standard}\quad {error}} = {\sigma = \sqrt{\frac{{\sum\limits^{1arrow n}x^{2}} - ( {\sum\limits^{1arrow n}x_{n}} )^{2}}{n^{2}}}}$

The following table showes the average values and the correspondingstandard errors for 5 different times of degradation of the films by H.insolens complex.

Cellulose I films Cellulose III_(I) films Equivalent glucose Equivalentglucose solubilised (mg/100 ml) solubilised (mg/100 ml) Time StandardTime Standard (h) Average error (h) Average error 0 0   0.46 0 0   0.261 1.54 0.57 1 1.47 0.43 2 3.07 1.53 2 2.79 1.14 4 6.89 1.86 4 7.77 1.256 13.29  1.88 6 13.07  1.44

The kinetics of degradation of the films of cellulose I and celluloseIII_(I) are very similar. This behaviour is in agreement with thekinetics experiments performed in the case of suspensions. Thereproducibility of the films deposition have been tested by an indirectmethod that include several experimental steps such as dilution andchemistry. Consequently, the resulting calculated standard errorscomprised the errors on the films formation and others experimentalerrors as well.

Example 11

Enzymatic degradation of labelled films was tested. Films of MFC wereobtained by drying 100 μl of cellulose suspensions (1 mg/ml) at 37° C.per well of a 96 well microtiter plates. The tested MFC was that whichallowed the maximum release of fluorescence after incubation withcellulases in suspension. The MFC used for making the film wasaccordingly grafted by 4 repeated labelling steps with 60 mg DTAF in thecase of cellulose I and 3 grafting steps in the case of celluloseIII_(I).

The reactivity of the films were assayed towards EG V and EG VIactivities. 10 μl of the enzymes (0.1 mg/ml) were deposited in the wellscontaining 200 μl of 50 mM phosphate buffer pH 6.5. The microtiterplates were kept at 37° C. At various times of degradation, 8 sampleswere collected and diluted 8 times and 200 μl of these dilutions wereanalysed by spectrofluorometry. The average and the standard errorvalues calculated from the fluorometry data recorded on the labelled MFCI and cellulose III_(I) shows from the following tables:

No enzyme EG V Fluorescence (R.I.) Fluorescence (R.I.) Cellulose IStandard Standard Time (h) Average error Average error 0 148.07 15.52145.93 28.09 1 175.02 17.12 260.2  43.15 2 214.42 20.98 361.36 65.93 3221.43 29.65 424.11 79.26 4 230.70 31.68 405.70 78.96 5 225.84 32.11416.98 91.41 No enzyme EG V EG VI Fluorescence Fluorescence FluorescenceCellulose (R.I.) (R.I.) (R.I.) III Aver- Standard Standard Standard Time(h) age error Average error Average error 1 62.03 8.50 52.93 16.33 65.257.94 2 81.14 10.70 325.52 67.08 237.60 34.88 3 86.45 14.047 539.80115.60 385.31 48.28 4 85.34 14.86 517.90 136.20 441.46 70.47 5 88.3119.13 544.62 137.12 481.05 84.57 6 87.45 18.15 567.90 150.10 427.07137.99

For both the cellulose systems and whatever the endoglucanase tested,the fluorescence release seems to occur according the same pattern. Thefluorescence increase linearly until a maximum which was reached after 3to 4 hours incubation with enzyme. When no enzyme was present in thereaction medium, the maximum intensity of the non specific fluorescenceis observed more quickly, usually in less than 2 hours.

Concerning the reproducibility of the kinetics followed by thefluorescence release, it is observed that this system allow within theexperimental errors to evidence minute amount of endoglucanes in lessthan 1 hour. It is important to notice, that the standard errorsrecorded is the sum of experimental errors. Some of these have occurredcertainly after the numerous dilutions necessary to scale down thestrong fluorescence intensity with the spectrofluorometer sensitivity.

The labelled cellulose substrates do not have the same behaviours.Indeed, in the case of EG V, it is observed that the use of celluloseIII_(I) instead of cellulose I allows a gain of fluorescence release ofa factor 2.5.

Example 12

Experiments using yeast extracts were performed on films of labelledMFC. The results presented in FIG. 10 were obtained by incubating filmsof cellulose III_(I) with 200 μl of 50 mM phosphate buffer pH 6.5 towhich was added 50 μl of yeast extract. Despite strong quenching, it waspossible to follow the increase of fluorescence at various time ofdegradation when the yeast extract containing active EG VI. Thesolubilisation of the fluorescent probes increased until a maximum wasreached in 3 hours. The difference in fluorescence intensities betweenthe yeast extract containing the active EG VI and one having a mutatedinactive EG VI, suggests that the EG VI activity could be reasonablydetected in less than 2 hours.

Example 13

DTAF was attached or grafted on MFC in a multi-step procedure asdescribed in example 7: 60 mg of DTAF was added to 10 ml of cellulosesuspension (10 mg/ml) in 0.2M NaOH. The mixture was stirred for 24 hoursat room temperature. Then, the specimens were washed extensively bycentrifugation with distilled water. The labelling was performed severaltime and the final cellulose suspensions were stored at 4° C.

Example 14

The labelled of microfibrillated cotton cellulose of example 13 wastested towards EG V and EG VI. 600 μl of grafted cellulose suspension(100 μg/100 μl) in 50 mM phosphate buffer at pH 6.5 were mixed with 20μl of enzyme (1 mg/ml) or 20 μl of water as control or standard. Thehydrolysis was conducted for 4 hours at 37° C. without agitation.Supernatants of the respective centrifuged test solutions were analysedby the ferricyanide method and by spectrofluorometry. FIGS. 11 and 12shows the release of fluorescence in samples diluted 16 times as afunction of the number of labelling steps when the labelled ofmicrofibrillated cotton cellulose were incubated with EG V and EG VIrespectively. In both cases, the variation of fluorescence follows thesame behaviours as that observed for bacterial cellulose. Indeed, theamount of fluorescent probes increase with the number of labelling stepsuntil a maximum reached for the fourth grafting steps. Beyond that, thedetected fluorescence intensity decreased for the most graftedcellulose. The number of steps that are necessary to obtain a maximum offluorescence release in the case of microfibrillated cotton cellulose isidentical to what we previously observed with bacterial cellulose I.

Example 15

Films of microfibrillated cotton cellulose was prepared. The cellulosefilms were obtained according to the same procedure described in example9: 100 μl of cellulose suspensions (1 mg/ml) per well of microtiterplates were dried overnight at 37° C.

Example 16

The reactivity of films of unlabelled microfibrillated cotton cellulosewere tested towards the H. insolens complex. Each well of themicro-titer plates were filled with 200 μl of 50 mM phosphate buffer atpH 6.5 followed by the addition of 20 μl of H. insolens complex (1mg/ml). The mixtures were kept at 37° C.

At various time of incubation, 8 samples of 100 μl were collected andthe amount of solubilised reducing sugars produced were measured by theferricyanide method. Averages and standard errors values calculated fromthe sets of data are shown in the following table:

Equivalent glucose solubilised (mg/100 ml) Time Standard (hours) Averageerror 0 0   0.28 1 2.21 0.66 2 5.13 1.27 4 9.87 1.75 6 17.66  1.19

The degradation kinetics, the standard error of the overall experimentincluding films deposition, dilution, and the reducing sugars test wasin agreement with a good reproducibility of the film deposition as itwas observed previously for the bacterial cellulose I and III_(I).

Example 17

The reactivity of films towards enzymes was assayed with the labelledmicrofibrillated cotton cellulose which allowed the maximum release offluorescence after incubation with enzymes. This labelled cottoncellulose was obtained after 4 repeated labelling steps with 60 mg DTAFin 0.2 M NaOH.

The degradation of the films was performed by adding 10 μl of EG V andEG VI (0.1 mg/ml) to 200 μl of 50 mM phosphate buffer pH 6.5 depositedin the wells of the microtiter plates. The samples were kept at 37° C.At various degradation time, 8 samples were collected and diluted 8times. 200 μl of these diluted solutions were analysed byspectrofluorometry. The average and the standard error values calculatedfrom the recorded data are shown in the following table:

No enzyme EG V EG VI Fluorescence Fluorescence Fluorescence (R.I.)(R.I.) (R.I.) Time Standard Standard Standard (hours) average errorAverage error Average error 0 206.08 15.76 197.60 24.07 232.47 29.66 1245.22 17.34 325.12 52.44 328.25 56.06 2 287.01 25.85 454.82 87.93457.45 73.18 3 289.89 30.56 549.99 95.97 526.94 53.86 4 277.01 22.51547.62 117.77 539.83 52.38 5 271.00 33.43 573.95 121.79 567.67 66.67

The degradation pattern of the labelled cellulose films were verysimilar to those that were obtained with bacterial cellulose I in termof intensity of the fluorescence and kinetics of solubilisation of theprobes. Consequently, as for bacterial cellulose it seems reasonablethat the endoglucanase activities could be detected in less than 2 hourswith the use of such labelled cellulose films.

Example 18

Haemoglobin was labelled with Fluorescein-isothiocyanate; Isomer I(FITC). 17.500 g bovine hemoglobin (Sigma H-2625) was dissolved in 600mL 0.25 M sodium-buffer (pH=9.0). 75 mg FITC (Sigma F-1522) dissolved in250 mL 0.25 M sodium-buffer (pH=9.0) was added drop-wise over 10 minutesunder vigorous stirring. The mixture was allowed to react in dark atroom temperature for 1 hour. Excess of FITC was removed by ultrafiltration on a Filtron Amicon RA2000 against PBS-buffer (containing80.0 g NaCl (Merck 6404), 2.0 g KCl (Merck 4936), 10.4 g K₂HPO₄ (Mecrk5101), and 3.17 g KH₂PO₄ (Merck 4873) in 10.00 L miliQ water; pH=7.2 ).

Example 19

Galactomannan (Locust bean gum) was labelled withFluorescein-5-thiosemicarbazide. 3.0032 g Galactomannan (Sigma G-0753)dissolved in 250 mL miliQ water was oxidised at room temperature for 48hours using Galactoseoxidase (Cibrina candolleana 8637/F9700806). Theoxidation was followed by light-absobance (Abs₄₁₀), after treating asmall sample with a few drops of a PHBAH-reagent (containing 150 mgp-hydroxybenzosyrehydrazid, and 500 mg Potassium-sodium-tartrate in 10.0mL 2% NaOH-solution) at 95° C. for 5 minutes. The enzyme was inactivatedby heating the mixture to 90° C. for 5 min. 75.2 mgfluorescein-5-thiosemicarbazide (Molecular Probes F-121) dissolved in 2mL DMF was added, and the mixture was allowed to react at roomtemperature in dark for 48 hours. The labelled polymer was precipitatedin 400 mL MeOH and was subsequently washed using EtOH until thesupernatant no longer contained probe. The labelled polymer wasre-dissolved in water and freeze-dried. Produced amount: 2.112 g.

Example 20

A new batch of Bacterial Cellulose I was prepared: The contents of 3cans of Nata de Coco containing approximately 900 g wet bacterialcellulose from Acetobacter Xylium in cubes was washed in 10 Ldemineralised water. The cubes were then washed in 3 L 1% NaOH solution.The soda was changed twice every day for 5 days. The cubes were finallywashed in 3 L demineralised water. The water was changed twice every dayfor 5 days. The cubes were homogenised in a warring blender and dialysedagainst demineralised water (Cut-off 12-14000) for 4 days.

Example 21

Cellulose films containing labelled haemoglobin or labelledgalactomannan were prepared: Suspended bacterial cellulose of example 20(1 mg/ml) was mixed with 500 μg/ml fluorescein labelled haemoglobin or50 μg/ml fluorescein labelled galactomannan (Locust bean gumgalactomannan) and 25 μg/ml Keltrol T xanthan (Kelco, Chicago, USA)prior to being dispersed and microfibrillated using a Polytron PT 3000(Kinematica, Switzerland) for 3 minutes at 10.000 rpm. 100 μl, 20 μl,and 3 μl of this mixture was added to each well of 96, 384, and 1536well plates respectively, and dried over night at 37° C. The microtiterplates was 96 well (cat. # 442404) and 384 well (cat. # 464718) plateswith Maxisorp™ surface obtainable from NUNC, Denmark and the 1536 wellplates were obtained from Greiner labortechnik, Germany, cat. # 782101.

Enzymes were detected using the prepared film of example 21. Allenzymatic detections were conducted in 50 mM HEPES pH 8.0 with 1 mMCaCl₂. For detections conducted in 96, 384 and 1536 wells microtiterplates 165 μl, 80 μl and 8 μl of the diluted enzyme were added to eachwell, respectively. The reaction was incubated at 40° C. for 40 minutesat 700 rpm in a Thermostar (BMG, Germany). When 96, 384 and 1536 wellsplates were applied samples of 100 μl, 60 μl and 4 μl were transferred,respectively, after the incubation to a new black microtiter andanalysed for fluorescence intensity on a Polarstar Galaxy (BMG, Germany)equipped with the appropriate light guides. Black 96, 384 and 1536 wellsplates were obtained from Bibby Sterilin, England, cat # 611F96BK; NUNC,Denmark, cat # 264556; and Greiner labortechnik cat # 782076,respectively.

Example 22

Protease activity of two different proteases was detected using thefilms containing labelled haemoglobin and the detection method ofexample 21. In the following table the amount of removed labelledhaemoglobin in % w/w are shown for 96, 384 and 1536 well plates:

96 well 384 well 1536 well plate plate plate Savinase ® 0.25 μg/ml 42%47% ND  0.5 μg/ml 50% 66% 42%  1.5 μg/ml 100%  100%  100%  C-component0.25 μg/ml 16% 21% ND  0.5 μg/ml 10% 12% ND  1.5 μg/ml 12% 14% ND

Savinase® is a commercially available protease from Novo Nordisk A/S,while component C is the glutamic acid specific protease described inKakudo S. et al.; Purification, Characterization, Cloning and Expressionof a Glutamic acid-specific Protease from Bacillus lichiniformis ATCC14580; J. Biol. Chem.; 1992; vol. 267; No. 33; pp 23782-23788. ND meansnot determined.

As shown the removal fluorescent Labelled haemoglobin in the 96, 384 and1534 well plate format corresponds very well demonstrating thatdetection of enzymes may be scaled down to very small volumes.

Example 23

Mannanase activity of four different mannanases was detected using thefilms containing xanthan and labelled galactomannan and the detectionmethod of example 21. In the following table the amount of removedlabelled galactomannan in % w/w are shown for 96 well plates relative toBXM 3 (10 μg/ml BXM 3=100%):

0.2 μg/ml 10 μg/ml BXM 1  9% 36% BXM 3 65% 100%  BXM 5 40% 92% BXM 7  0%21% BXM1, BXM5 and BXM7 is described in the international patentapplication PCT/DK99/00314.

The results shows that using a cellulose film containing labelledgalactomannan different mannanases may be ranked and BXM 3 may beselected as showing the best performance.

Example 24

A comparison of detecting the mannanase activity by the method ofexample 21 and detecting mannanase activity using textile swatches weremade: In order to dye textile swatches with fluorescently labelledgalactomannan, the textile was submerged into a aqueous solution of0.225 g/l unlabelled Locust bean gum (Sigma, USA), 0.025 g/l fluoresceinlabelled Locust bean gum and 0.125 g/l Keltrol T xanthan (Kelco,Chicago, USA). The textile was then put through a roller in order toremove any surplus of dye solution and subsequently air dried over nightin the dark. Finally the dyed textile was rinsed twice for 1 hour in 141 distilled water with 2 g/l detergent and air dried in the dark.

Detection of mannanase activity using textile swatches was done byincubating solutions of mannanase (BXM 3) with the labelled textileswatch for 40 minutes at 40° C., while shaking at 700 rpm. Subsequently,the solution was aspirated applying a plate washer (EL 403H, Bio-TekInstruments, Vermont, USA) and the fluorescence of the labelledgalactomannan remaining in the textile was measured by the use of aPolarstar Galaxy (BMG, Germany).

Detection of Mannanase activity using labelled cellulose film was doneusing the films containing xanthan and labelled galactomannan and thedetection method of example 21 with the exception that the fluorescenceof the labelled galactomannan remaining in the film was measured.

The results of the comparison is shown in the following table:

μ/ml Textile Bacterial Cellulose BXM 3 (% change) (% change) 15.0  99.8100   5.0 110.4  106.0 2.5 91.5 90.3 1.0 51.2 66.7 0.2 17.3 28.8 0.0 0 0 

The results shows that the change of fluorescence versus concentrationof BXM 3 mannanase is similar for both the textile and the cellulosefilm. Accordingly the removal of substrate from a cellulose filmsimulates very well the removal of substrate from a textile and that useof a film of MFC in microscale containers may replace textile whendetecting enzymes. The standard derivation of the textile and bacterialcellulose film assay is 4-8% and 2-8%, respectively, based on 4measurements, proving that detecting using a cellulose film is morereproducible.

Example 25

Dual probe assay for enzyme specificity.

This experiment was conducted to show that a cellulose film can beprepared incorporating two different enzyme substrates towards whichdifferent enzymes have different specificity. By mixing two differentsubstrates, each labelled with a probe with unique spectral properties,one can use the ratio of the signals to categorize the specificity of anenzyme sample for the two substrates.

To illustrate this concept using cellulases two substrates wereprepared: Carboxymethylcellulose labelled with eosin (CMC-E) andbacterial cellulose labelled with fluorescein (BC-F). A film wasprepared in micro titer plate wells with a mixture of these twosubstrates, and the film was incubated with either of two differentcellulases: Endoglucanase I or Endoglucanase V from H.insolens (bothcloned and expressed in A.oryzae as described earlier). These twoenzymes are known to have different substrate specificities.

Experimental

BC-F was prepared as described previously.

CMC-E was prepared by the following procedure: 1,005 g CMC was dissolvedin 50 mL water and pH was adjusted to 5,9 on 0.1 N NaOH. 48.9 mg5-aminoeosin dissolved in 2 mL DMF was added. 1.24 g EDAC was added insmall portions over 1 h. The reaction mixture was stirred overnight atroom temperature. The product was precipitated in a mixture of 15 mLMeOH and 500 mL EtOH. The labelled polymer was washed in EtOH andfreeze-dried 10 microliter CMC-E (0,5 mg/ml) was mixed with 100microliter BC-F (1 mg/ml) in each well of a 96-well plate and incubatedat 50° C. overnight to form the dual labelled film.

Solutions of EG I and of EG V at concentrations of 0; 62,5; 125; and 250mg/L were prepared.

25 microliter enzyme solution and 200 microliter buffer (0,05 M tris, pH7,6) was incubated in each well for 2 h at 37° C.

25 microliter of the supernatant of each well was taken as samples andwas diluted with 200 microliter buffer and the fluorescence intensity at515/555 nm (for eosin) and 485/520 nm (for fluorescein) was measuredusing a Polarstar fluorimeter. Each value was corrected for the averagefluorescence intensity of the blank samples, and the ratio of thecorrected eosin and fluorescein fluorescence intensities was calculatedas an indication of the enzyme specificity.

Results

Conc. (mg/L) EGI EGI EGV EGV Eosin fluorescence intensity (corrected;arbitrary units) 0 0 ± 626 62.5 1822 2436 3753 3268 125 2489 2847 49284382 250 2141 2672 5406 5312 Fluorescein fluorescence intensity(corrected; arbitrary units) 0 0 ± 65 62.5 1450 1720 6073 5666 125 18562219 7180 6301 250 2065 2897 9627 9882 Ratio of corrected fluor.intensities (eosin/fluorescein) 0 N.D. 62.5 0.80 0.71 1.62 1.73 125 0.750.78 1.46 1.44 250 0.96 1.08 1.78 1.86

It can be seen from the results, that the EGV enzyme in all cases give amuch higher E/F ratio, indicating the separate specificity of thisenzyme. As EGV and EGI are both commercial enzymes that each have aseparate function in detergents, this assay format can be useful torapidly determine if cellulase enzymes have EGI-like or EGV-likesubstrate specificity.

Example 26

Use of bacterial cellulose films for assaying protein degradation fromsolid surfaces

Fluorescently labelled haemoglobin was mixed with bacterial cellulose,and a cellulose film was prepared in the wells of polystyrene 96-wellmicrotiter plates. The cellulose films were dried and used indose-response wash performance experiments with protease. The “stainremoval” ability of laundry detergent with Savinase® protease (aconventional detergent protease) was tested.

Labelling of Heamoglobin

Fluorescein-5-isothiocyante ‘isomer I’ (FITC; Molecular Probes F-143)was covalently coupled to bovine haemoglobin (Sigma H-2625, lot.125H9310) by dissolving 15.4 g of the protein in 600 ml 0.25 M NaHCO₃ pH9.0, and 156.8 mg FITC in 250 ml 0.25 M NaHCO₃ pH 9.0. The two solutionswere mixed and stirred in darkness for 60 min at room temperature.Unbound FITC was removed by gel filtration on a 4 1 Sephadex 25 column(Amersham Pharmacia Biotech). The collected 880 ml was supplemented withglycerol to a final concentration of 50% (w/v). 0.1% (w/w) 50% glutaricaldehyde (Merck 814393) was added and the mixture was stirred for 1 h atroom temperature.

Bacterial Cellulose

Bacterial cellulose (BC) was obtained from Nata de Coco (Del Monte) bywashing the cubes in water followed by 5 overnight washes in 1% NaOH,and 6 overnight washes in water. The cubes were subsequently homogenisedin a blender and dialysed against mili Q water (12-14,000 cut-off). Thefinal product was in a concentration of 1 g solids per litre water.

Preparation of Cellulose Film With Labelled Haemoglobin

The prepared FITC-haemoglobin and BC were mixed in ratios Heamoglobin:BCof 1:10, 1:4 and 1:2 and films were prepared by dispensing thesemixtures in wells of polystyrene microtiter plates (Nunc 269620). Filmswere formed by drying the dispensed mixtures overnight at 50° C.

Samples of different concentration of purified Savinase® were preparedin water and dissolved in 6 g/l of a commercial detergent. A total of165 μl protease in detergent was added per well. The plates were shakenfor 30 min at room temperature and 100 μl of the wash supernatants weretaken as samples and transferred to black microtiter plates (Sterilin611F96BK). Fluorescence (excitation at 485 nm, emission at 520 nm) ofthe supernatants was measured in a spectrofluorometer (BMG Polarstar).

Results

Fluorescence intensity (arbitrary units) Heamoglobin:BC 1:2 1:4 1:10 μgprotease/ml 0 20745 14848  6086   0.1 25687 16773  6437   0.25 2865920279  8210   0.5 32892 25093 10779 1 35339 30037 14336 2 38813 3537018134 3 42860 38996 18118 5 47854 41867 20472

At the selected conditions, the fluorescence of the wash supernatantsincreased with enzyme dosage reflecting increased amounts of releasedhaemoglobin, while the level of fluorescence increased with theincreased amount of labelled haemoglobing in the film. The example alsoshow that the performance of an enzyme which works well in a realcleaning application can be evaluated in a test system of the inventionusing a cellulose film in stead of real textile.

What is claimed is:
 1. A method for screening for an active comprisingcontacting a sample containing the active with a cellulose filmcomprising microfibrillated cellulose and detecting an interactionbetween the cellulose film and active.
 2. The method of claim 1 furthercomprising the steps of (a) depositing a cellulose film comprisingmicrofibrillated cellulose on at least one inner surface of a container,(b) adding the active dissolved or dispersed in a liquid to the film,(c) Incubating the film with the active and (d) monitoring theinteraction between the active and the cellulose film, preferably bymeasuring a compound which have been released from the film by theinteraction.
 3. The method of claim 1, wherein the active is selectedfrom the group consisting of biological compounds, inorganic detersivecompounds and organic detersive compounds.
 4. The method of claim 3,wherein the biological compound is an enzyme.
 5. A method for screeningfor a nucleic acid sequence encoding a biological compound, the methodcomprising: (a) expressing the nucleic acid sequence in an expressionsystem, so as to produce the biological compound, (b) contacting thebiological compound with a cellulose film, (c) measuring an interactionbetween the biological compound and the cellulose film and (d) selectingexpression systems for which a detectable interaction occurred andrecovering the nucleic acid sequence.
 6. The method of claim 5, whereinthe expression system is an in vitro coupled translation/transcriptionsystem.
 7. The method of claim 5, wherein the expression system is acellular expression system.
 8. The method of claim 7, wherein thecellular expression system is a wild type cell.
 9. A method forproducing a biological compound comprising: a) contacting a cellulosefilm with a population of cells or in vitro expression systemscomprising a gene library that expresses at least one biologicalcompound of interest, (b) identifying in the population, a cell or invitro expression system which expresses a biological compound thatinteracts with the cellulose film; (c) selecting the cell or In vitroexpression system producing the biological compound, (d) identifying anucleic acid sequence encoding the biological compound, (e) cultivatinga cell comprising a nucleic acid sequence encoding the biologicalcompound so as to produce the biological compound and, (f) recoveringthe biological compound.
 10. A modified cellulose film comprisingmicrofibrillated cellulose, wherein the film further comprises asubstance attached to the cellulose film.
 11. The cellulose film ofclaim 10, wherein the substance is attached to the cellulose film bycovalent bonds, by ionic bonds or by hydrogen bonds.
 12. The cellulosefilm of claim 11, wherein the substance is attached onto the surface ofthe film after formation of the film.
 13. The cellulose film of claim10, wherein the compound is selected from the group consisting of a dye,a radioactive compound, a non cellulose substrate andnon-microfibrillated cellulose substrate.
 14. The cellulose film ofclaims 13, wherein the dye is selected from the group consisting oflight absorbing dyes and fluorescent dyes.
 15. The cellulose film ofclaims 14, wherein the dye is a fluorescent dye.
 16. The cellulose filmof claims 13, wherein the radioactive compound comprises an isotopeselected from the group consisting of S35, P32, H3 and I125.
 17. Amethod preparing a cellulose film of claim 10 comprising preparing asuspension of microfibrillated cellulose, sedimenting themicrofibrillated cellulose as a film onto a surface and contacting themicrofibrillated cellulose with a substance before, during or after theformation of the film.
 18. A test container for biological screeningcomprising at least one surface coated with a cellulose film.
 19. Themethod according to claim 10, wherein the cellulose film comprises asubstance attached to the cellulose film and the biological compoundinteracts with the substance.